Figure 3.

Dr1 associates with Brf1. (A) Schematic model of how the pol III initiation complex was predicted to be disrupted by Dr1 (21). (B) Brf1 was in vitro translated and radio-labelled with ³⁵S, mixed with HeLa nuclear extracts and then used in co-IP experiments with Dr1 1162 antiserum and pre-immune serum (PI). (C) Antiserum for Dr1 (1162) was used to co-immunoprecipitate endogenous Brf1 from HeLa nuclear extracts. 1162 pre-immune serum (PI) was used as control. (D) Overexpression of Brf1 results in increased Dr1 crosslinking at pol III-transcribed genes. CHO cells, stably transfected with an inducible Brf1 TET-OFF expression system (23), were induced to express Brf1 for 48 h before harvesting. ChIP experiments were performed using antibodies against TBP, Brf1 and Dr1 (1162). Pre-immune (PI) serum and rabbit IgGs were used as negative controls. The ‘+’ denotes induction of Brf1 compared to the uninduced ‘−’ control. (E) Western blot demonstrating that Dr1 protein levels do not increase after induction of Brf1 in the CHO cell Brf1 inducible system. (F) DNA recovered from ChIPs carried out using rabbit IgGs or antibodies against Brf1 and TBP was used for a second ChIP with preimmune serum or sera against Dr1 and pol III, as indicated. Input lanes show 10% of total input employed for the secondary ChIP.